Neurobiology of Disease
Genetic Deletion of Cdc42 Reveals a Crucial Role for Astrocyte
Recruitment to the Injury Site In Vitro and In Vivo
Stefanie Robel,1,2* Sophia Bardehle,1,2* Alexandra Lepier,1 Cord
Brakebusch,3 and Magdalena Gotz1,2
1Physiological Genomics, Institute of Physiology,
Ludwig-Maximilians University Munchen, 80336 Munchen, Germany,
2Institute for Stem Cell Research, HelmholtzZentrum Munchen, 85764
Neuherberg, Germany, and 3Biotech Research and Innovation Centre,
University of Copenhagen, 2200 Copenhagen, Denmark
It is generally suggested that astrocytes play important
restorative functions after brain injury, yet little is known
regarding their recruitment to sites of injury, despite numerous in
vitro experiments investigating astrocyte polarity. Here, we
genetically manipulated one of the proposed key signals, the small
RhoGTPase Cdc42, selectively in mouse astrocytes in vitro and in
vivo. We used an in vitro scratch assay as a minimal wounding model
and found that astrocytes lacking Cdc42 (Cdc42) were still able to
form protrusions, although in a nonoriented way. Consequently, they
failed to migrate in a directed manner toward the scratch. When
animals were injured in vivo through a stab wound, Cdc42 astrocytes
developed protrusions properly oriented toward the lesion, but the
number of astrocytes recruited to the lesion site was significantly
reduced. Surprisingly, however, lesions in Cdc42 animals, harboring
fewer astrocytes contained significantly higher numbers of
microglial cells than controls. These data suggest that impaired
recruitment of astrocytes to sites of injury has a profound and
unexpected effect on microglia recruitment.
Introduction Astrocytes play crucial roles in the adult brain, yet
the molecular mechanisms governing their specific functions are
still poorly understood. Throughout the brain astrocytes occupy
distinct ter- ritories (Bushong et al., 2002; Ogata and Kosaka,
2002), where they perform various functions including the
regulation of blood flow in response to neural activity (Iadecola
and Nedergaard, 2007; Schummers et al., 2008), requiring contact of
their endfeet to blood vessels. Astrocytes are polarized toward the
basement membrane around blood vessels and target proteins, such as
aquaporin-4 to their endfeet (Bragg et al., 2006). If this
interface fails to form properly, as is the case following a loss
of 1-
integrins, there results a mild reactive gliosis with all hallmarks
of reactive astrogliosis except proliferation (Robel et al., 2009),
highlighting the importance of astrocyte polarity. However, little
is known about the role of astrocyte polarity after brain injury in
vivo.
The reaction of astrocytes to brain injury presents as reactive
astrogliosis that ranges from wound closure through astrocyte
dedifferentiation, to scar formation (Ridet et al., 1997; Silver
and Steindler, 2009; Sofroniew and Vinters, 2010). Astrocyte
activa- tion is characterized by hypertrophy and upregulation of
many proteins, including the intermediate filaments vimentin and
glial fibrillary acidic protein (GFAP), and proteins expressed at
earlier developmental stages, such as nestin, Tenascin C or
phosphacan (Buffo et al., 2008; Sirko et al., 2009). Interestingly,
following severe injury, a large fraction of reactive astrocytes
proliferate and some even regain stem cell potential (Buffo et al.,
2008; Robel et al., 2011). While this dedifferentiation may be
considered bene- ficial, reactive astrocytes also upregulate
various cell surface mol- ecules, e.g., chondroitin sulfate
proteoglycans, and participate in scar formation and inhibition of
axon growth across this region (Reier and Houle, 1988; Busch and
Silver, 2007). Thus, astrocytes perform numerous functions in
response to injury, partially dif- fering depending on the type and
size of injury (Pekny and Pekna, 2004; Sofroniew, 2009).
A key aspect common to many injuries is the increase in as- trocyte
number at the injury site, which has been suggested to be a result
of oriented migration and proliferation (Okada et al., 2006; Buffo
et al., 2008; Simon et al., 2011). Given that the presence of
astrocytes at the injury site is functionally impor- tant
(Sofroniew, 2009), it is critical to understand the molecular
machinery governing astrocyte polarity and recruitment to the
injury site in vivo. The small RhoGTPase Cdc42 has emerged as
a
Received May 31, 2011; revised July 7, 2011; accepted July 11,
2011. Author contributions: S.R. and M.G. designed research; S.R.
and S.B. performed research; A.L. and C.B. contrib-
uted unpublished reagents/analytic tools; S.R. and S.B. analyzed
data; S.R., S.B., and M.G. wrote the paper. This research was
supported by grants from the Deutsche Forschungsgemeinschaft (DFG
GO 640/7-1, 8-1, 9-1;
SPP-1048), including the excellence cluster Center for Integrated
Protein Science Munich, the European Community (Integrated Project
EuTRACC, Grant no. LSHG-CT-2007-037445), the SFB 596 and 870, and
the Bundesministerium fur Bildung und Forschung (Grants 01GN0503
and FKZ: 01 GN 0820); and the Helmholtz Association in the frame-
work of the “Helmholtz Alliance for Mental Health in Ageing
Society” (HELMA), the Impulse & Networking Fund of the
Helmholtz Association (HA-215), the Virtual Institute for
Neurodegeneration & Ageing (VH-VI-252), as well as by the
Bavarian research network “ForNeuroCell.” We thank foremost the
Graduate School of Systemic Neuroscience for financial support of
the time-lapse video microscope, without which this study would not
have been possible; Alexander Pfeifer (University of Bonn) for the
EGFP and Cre-IRES-EGFP lentiviral plasmids; Christian Naumann for
cloning the tdTomato and tdTomato-IRES-Cre lentiviral plasmids, as
well as for establishment of the time-lapse imaging; Silvia
Cappello, Swetlana Sirko, Susan Buckingham, Vishnu Cuddapah, and
Harald Sontheimer for critical comments on the manuscript; and Gabi
Jager, Simone Bauer, Andrea Steiner-Mezzadri, Tatiana Simon-Ebert,
and Rebecca Krebs for excellent technical assistance.
*S.R. and S.B. contributed equally to this work. S. Robel’s current
address: Department of Neurobiology and Center for Glial Biology in
Medicine, University of
Alabama at Birmingham, Birmingham, AL 35294. Correspondence should
be addressed to Magdalena Gotz, Physiological Genomics,
Ludwig-Maximilians University
Munchen, Pettenkoferstrasse 12, 80336 Munchen, Germany. E-mail:
[email protected].
DOI:10.1523/JNEUROSCI.2696-11.2011
Copyright © 2011 the authors 0270-6474/11/3112471-12$15.00/0
The Journal of Neuroscience, August 31, 2011 • 31(35):12471–12482 •
12471
key regulator of polarization, influencing directional migration in
cultured fibroblasts and astrocytes (Nobes and Hall, 1999;
Etienne-Manneville and Hall, 2001, 2003). However, these re- sults
were obtained using dominant-negative forms of Cdc42, and genetic
deletion of Cdc42 in fibroblasts revealed discrepan- cies in
polarization effects and directed migration (Czuchra et al., 2005).
This is probably due to inhibition of several other mem- bers of
the RhoGTPase family by dominant-negative Cdc42 (Czuchra et al.,
2005). Therefore, we set out to determine the role of astrocyte
polarity by investigating the Cdc42 function in astro- cytes in
vitro and in vivo using genetic tools to delete Cdc42.
Materials and Methods Animals and surgical procedures
C57BL/6J//129/Sv-Cdc42 mice carrying alleles for Cdc42 flanked by
loxP sites (Wu et al., 2006) were mated to mice expressing a
Cre-recombinase estrogen receptor fusion protein in the GLAST locus
(Mori et al., 2006). To label recombined cells, the CAG-CAT-EGFP
reporter line, expressing the CMV (-actin promoter) and the loxP
flanked chloramphenicol acetyltransferase (CAT) gene upstream of
the EGFP cassette (Nakamura et al., 2006) have been used. Mice of
either sex were included in the analysis.
All animal procedures were performed in accordance with the
Policies on the Use of Animals and Humans in Neuroscience Research,
revised and approved by the Society of Neuroscience and the state
of Bavaria under license number 55.2-1-54-2531-23/04 or
55.2-1-54-2531-144-07. Tamoxifen was administered as described
previously (Mori et al., 2006). For stab wound injury, animals were
deeply anesthetized and fixed in a stereotactic frame. Stab wounds
were placed into the somatosensory cor- tex of the right hemisphere
(1.5–2 mm long, 0.2 mm wide and 0.5– 0.6 mm deep).
Histological procedures Adult animals were deeply anesthetized and
transcardially perfused with PBS followed by 4% PFA in PBS (100
ml/animal). Brains were postfixed in the same fixative for at least
2 h to maximal overnight at 4°C, washed in PBS, and embedded in 4%
agarose for cutting 60 m vibratome sections.
For immunofluorescent labeling, sections were incubated overnight
at 4°C in PBS containing the first antibody, 0.5% Triton X-100 (TX)
and 10% normal goat serum (NGS), washed in PBS, and incubated for 2
h at room temperature in 0.5% TX and 10% NGS containing the
secondary antibody. After washing in PBS, sections were mounted on
glass slides and embedded in Aqua-Polymount and covered by a glass
coverslip.
Primary and secondary antibodies are listed in Table 1 and Table
2.
The terminal deoxynucleotidyl transferase dUTP nick end labeling
(TUNEL) assay was performed using an in situ cell death detection
kit (Roche) in accordance with the manufacturer’s
instructions.
The cresyl violet (Nissl) staining was performed as follows. Free-
floating vibratome sections were mounted and dried on glass slides
be- fore they were dehydrated and washed in xylene two times for 10
min to remove lipid-rich structures. After rehydration, sections
were stained in a 0.1% cresyl violet solution spiked with acetic
acid for 3 min, then washed, dehydrated, and cleared in xylene,
before they were embedded in Permount mounting medium and covered
by a glass coverslip.
Lentivectors and lentiviral preparation Lentiviral expression
plasmids contained the sequence for EGFP or Cre- IRES-EGFP under
the CMV promoter (Pfeifer et al., 2001). To avoid any differences
in expression levels of the fluorescent proteins, we modified these
constructs such that the red fluorescent protein tdTomato was
placed di- rectly behind the CMV promoter (LV-CMV-tdTomato and
LV-CMV- tdTomato-IRES-Cre), thus resulting in comparable signal
intensities. To generate the tdTomato-IRES-Cre vector, the
Cre-IRES-EGFP plasmid was digested using PstI and SalI to remove
the IRES-EGFP cassette. The IRES sequence was amplified with SpeI
linkers and placed in front of the Cre sequence into the SpeI
restriction site. The tdTomato sequence was then placed behind the
CMV promoter by digestion of the CMV-IRES-Cre vector using XbaI,
resulting in the lentiviral vector CMV-tomato-IRES-Cre. The
tdTomato control construct was generated by replacing the EGFP
cassette behind the CMV promoter with the sequence encoding
tdTomato.
The lentiviral expression plasmids described above, pCMVR8.91
packaging vector (Zufferey et al., 1997), and the pVSVG or pLCMV
envelope vector, were cotransfected into 293T cells for production
of lentiviral particles as described previously (Naldini et al.,
1996). Titers were determined on 293T cells, and for most
experiments, 8 10 6 viral particles were used per 500 l cell
suspension.
In vitro scratch injury assay The gray matter of the cerebral
cortex from 3– 4 postnatal mice (5–7 d old) was dissected and
mechanically dissociated in Hanks’ buffered saline solution
containing 10 mM HEPES. After washing in DMEM medium supplemented
with 10% fetal calf serum, 10 mM HEPES, and Penicillin/
Streptomycin, a single cell suspension was plated into T75 flasks
coated with poly-L-ornithine (PLO), and the medium was changed
every other day. After reaching confluence, progenitor cells on top
of the astrocyte monolayer were removed by thoroughly shaking the
cell culture flask, and astrocytes were passaged onto PLO-coated
coverslips or directly into PLO-coated 24-well plates for
time-lapse experiments. Astrocyte cul- tures were transduced by the
use of lentiviral particles during the splitting step after
resuspension of the cells, and directly plated at a density of
70,000 cells per well on plastic or 100,000 cells per well on glass
coverslips. Plates were placed into the incubator for 24 h at 37°C
and 5% CO2 before the medium replacement.
Table 1. First antibodies
Pretreatment incubation conditions Company/Source
Cdc42 Mouse IgG3 1:100 Santa Cruz Biotechnology (sc-8401) Cop1
(clone CM1A10) Mouse IgG1 1:100 Gift from J. E. Rothman, Yale
School
of Medicine, New Haven CT GFAP Mouse IgG1 1:1000 Sigma (G3898) GFAP
Rabbit 1:500 Dako/Invitrogen (Z0334) GFP Mouse IgG1 1:1000
Millipore (MAB3580) GFP Rabbit 1:1000 Invitrogen (A6455) GFP Chick
1:1000 Aves Lab (GFP-1020) Iba1 Rabbit 1:500 Wako (019-19741) NeuN
Mouse IgG1 1:100 Millipore (MAB377) -Tubulin Mouse IgG1 1:100 Sigma
(T5326) pan-Tubulin Rat 1:10 Gift from the Department of
Anatomy
and Cell Biology, Ludwig-Maximilians University München
RFP Rabbit 1:500 Millipore (AB3216) S100 Rabbit 1:100 Sigma (S2644)
S100 Mouse IgG1 1:500 Sigma (S2532)
Table 2. Secondary antibodies
Antibody Host species Label Dilutions Company
-Chick Goat Alexa Fluor 488 1:500 Invitrogen (A11039) -Chick Donkey
FITC 1:200 Dianova (703095155) -Rabbit Donkey Alexa Fluor 488 1:500
Invitrogen (A21206)
Cy3 1:500 Dianova (711165152) Alexa Fluor 594 1:500 Invitrogen
(A21207)
Goat Cy3 1:500 Dianova (111165144) Biotinylated 1:200 Vector
(BA-1000)
-Mouse IgG1 Goat Alexa Fluor 488 1:500 Invitrogen (A21121) Alexa
Fluor 594 1:500 Invitrogen (A21125) Biotinylated 1:200 South.B.
(1070-08)
-Mouse IgG2a Goat Alexa Fluor 488 1:500 Invitrogen (A-21131) Alexa
Fluor 594 1:500 Invitrogen (A-21135)
-Mouse IgG Goat Alexa Fluor 488 1:500 Invitrogen (A11029) Cy3 1:500
Dianova (115165166) Cy5 1:500 Dianova (115176072)
Donkey Alexa Fluor 594 1:500 Invitrogen (A-21203)
12472 • J. Neurosci., August 31, 2011 • 31(35):12471–12482 Robel et
al. • Cdc42 Is Important for Astrocyte Recruitment to Injury
Two weeks later, Cdc42 protein loss could be confirmed by immuno-
cytochemistry exclusively in cells transduced by the lentiviruses
contain- ing the Cre recombinase (see Fig. 2).
At the earliest, 2 weeks after viral transduction and 1 week after
con- fluence had been reached, in vitro scratch wound experiments
were started following a published protocol (Etienne-Manneville,
2006). Briefly, the confluent astrocyte monolayer was scratched
once from the left to the right wall of the well with a sterile 10
l plastic tip, resulting in a cell-free cleft 500 m wide.
For time-lapse experiments, primary astrocyte cultures were
prepared from cortices of postnatal Cdc42fl/fl mice (postnatal days
5–7, 3– 4 ani- mals per experiment) as described above. Two weeks
after transduction with tdTomato or tdTomato-IRES-Cre lentivirus,
scratch assay experi- ments combined with video time-lapse
microscopy were started. Before scratching the confluent monolayer,
Hoechst 33342 dye (Invitrogen) was added to the culture medium at a
final concentration of 1 g/ml, and incubated for 20 min at 37°C
with 5% CO2. Cells were washed twice with prewarmed culture medium
and scratched 2 h later. The plate was then placed into the
incubation chamber (37°C, 8% CO2) of an Observer Z1 (Zeiss)
fluorescence microscope. Imaging procedures were controlled using
AxioVision Rel. 4.7 software for acquisition of phase contrast im-
ages every 10 min, and fluorescence images once per hour, for 5 d
using a 20 objective and an AxioCam HR camera. To control for
potential effects due to Cre toxicity, we also transduced
astrocytes cultured from WT (C57BL/6) with the Cre-containing
virus, and found no signs of toxicity even 2 weeks after
transduction. Moreover, changes were ob- served neither in the
orientation of migration toward the scratch nor in the
tortuosity.
For analysis of fixed cells, cultures were either immunostained or
la- beled for actin filaments by phalloidin-Alexa Fluor 488
(Invitrogen) that was added to the secondary antibody
solution.
Data analysis Results are presented as the mean calculated between
different animals (at least three sec- tions per animal and three
animals for each time point unless stated differently) or between
independent cultures. The variation between animals or cultures is
depicted as SEM with one data point representing one animal.
Based on a Gaussian distribution, the data were statistically
analyzed by performing an unpaired t test. Means were considered
signif- icantly different according to the p value: *p 0.05, **p
0.01, and ***p 0.001. Calcula- tions and statistical analysis were
done with Excel and GraphPad Prism 3.0, 4.0, or 5.0. Means were
considered significantly different as indicated above.
Quantifications after stab wound in vivo. For analysis of astrocyte
protrusion formation after a stab wound injury in vivo, lesion size
and astrocyte proliferation were assessed using confo- cal images
taken with a Zeiss LSM700 confocal microscope. Length and width of
EGFP-positive cells, as well as the longest process toward the stab
wound, was measured using ZEN 2008/2009 software (Zeiss). To
analyze Nissl neuronal number in stab wound lesions, slices were
imaged using the Stereo Investigator (mbf Bioscience) software
interfaced with an upright Olympus BX-51 microscope. Traces were
drawn around regions of interest using a Plan Apo 10 objec- tive
corrected for bright-field observation. Counting was performed
using a Plan Apo 40 objective. The Stereo Investigator (mbf Biosci-
ence) software was also used to quantify micro- glia number in
confocal images taken using a Leica SP5 microscope.
Quantifications after scratch wound in vitro. Scratched astrocyte
cultures were stained for
microtubules that were then observed using a fluorescence
microscope (Olympus, BX61) with a 60 objective. Reorientation of
the centrosome [microtubule organizing center (MTOC)] in astrocyte
cultures after scratch wound was quantified by separating the area
around the nucleus into 4 equal quadrants that joined in the center
of the nucleus of the cell of interest. The quadrants were placed
with one quadrant facing the scratch and the median of each 90°
angle located either perpendicular or parallel to the scratch.
MTOCs were scored as reoriented when they were located in the
quadrant facing the scratch. Transduced cells in the first row
adjacent to the scratch that displayed one major protru- sion three
times longer than wide were scored as “protruding cells.” The data
were obtained from three independent preparations from different
litters. For each preparation and time point, two different
coverslips and at least 100 transduced cells per coverslip were
ana- lyzed and one coverslip was considered a single data
point.
Images from time-lapse video microscopy were assembled into a movie
and analyzed using the AxioVision Rel. 4.8 software (Zeiss).
Quantifications include virus-transduced cells that expressed the
red fluorescent protein tomato and lined the front of the scratch.
Hoechst labeled nuclei were tracked for three defined time points
(1, 3, 5 d). The individual tracking paths of every selected cell
were used to cal- culate the following parameters: mean velocity,
straight distance, to- tal distance (equals the path length) and
tortuosity (equates to the quotient of total distance and straight
distance). Protrusion number and transduced cell polarity was
assessed 12, 24, 48, and 120 h post- injury (p.i.) using red
fluorescence images. For protrusion turnover, the presence or
absence of each single protrusion was analyzed at a first and a
second time point for three different periods 0 –24 h p.i., 24 – 48
h p.i. and 48 –120 h p.i.
Figure 1. Astrocytes in vitro react to injury by polarization.
Postnatal astrocyte cultures were positive for GFAP (A) and/or S100
(B). After scratching, cells in the monolayer (C) reacted to the
injury and reduced the size of the gap over time (D–F ). Astrocytes
at the scratch formed an extension into the cell-free area (G–J ).
These protrusions were rich in tubulin and stabilized by the actin
cytoskeleton (G, H ). Centrosomes (MTOCs, arrowhead in I ) or Golgi
(J ) of polarized cells were reoriented facing the injury
area.
Robel et al. • Cdc42 Is Important for Astrocyte Recruitment to
Injury J. Neurosci., August 31, 2011 • 31(35):12471–12482 •
12473
Results Polarity of astrocytes after scratch injury in vitro
Previous studies used a scratch wound as- say after injection of
dominant-negative and constitutively active constructs to dem-
onstrate a role for the small RhoGTPase Cdc42 in astrocyte polarity
(Etienne- Manneville and Hall, 2001, 2003; Etienne- Manneville et
al., 2005). In the present study, we used the same assay to examine
the effects of Cdc42 genetic deletion in astrocytes. Toward this
aim, mouse as- trocytes were obtained from the postna- tal cerebral
cortex and grown to full confluence to allow for astrocyte matura-
tion. After 3– 4 weeks in culture, cells pre- sented with a flat
morphology and could be labeled with antibodies against the as-
trocyte proteins GFAP (Fig. 1A) and/or S100 (Fig. 1B). In
accordance with pre- vious observations (Etienne-Manneville, 2006),
after injuring the monolayer (Fig. 1C), astrocytes extended
processes toward the cell-free scratch region and subse- quently
migrated and populated the scratch over a 24 h period (Fig. 1D–F).
These scratch-oriented processes had tubulin-positive fibers in the
leading tips and were stabilized by the actin cytoskel- eton (Fig.
1G,H) at 24 h p.i. The forma- tion of protrusions was accompanied
by reorientation of both the centrosome (MTOC) labeled by -tubulin
(Fig. 1 I) and the Golgi apparatus labeled by Cop1 (Fig. 1 J)
toward the injury site, starting as early as 4 h after scratch in
some cells.
To examine Cdc42 expression in this culture model, astro- cytes
were stained for Cdc42 at different time points after monolayer
injury (Fig. 2A–C). Before and shortly after the scratch,
endogenous Cdc42 protein was found mainly around the nuclei of
astrocytes located at the scratch wound (Fig. 2A), whereas after 8
h, the protein relocalized toward the leading edge of astrocytes
facing the scratch (Fig. 2B,C). This is similar to what has been
reported after injecting constructs encoding Cdc42-GFP fusion
proteins (Etienne-Manneville and Hall, 2001; Osmani et al., 2010).
High-power magnification revealed that Cdc42 was enriched at the
tips of newly formed processes (Fig. 2C).
Deletion of Cdc42 reveals a crucial role in orientation of cells
toward scratch injury in vitro To investigate the role of Cdc42 in
astrocyte polarization, we used a genetic deletion designed to
avoid potential nonspecific effects of constitutively active and
dominant-negative con- structs. Postnatal mouse astrocytes
containing both alleles of the Cdc42 gene flanked by loxP sites (Wu
et al., 2006) were cultured and transduced with lentiviruses
containing the sequence for either Cre-IRES-EGFP/tdTomato-IRES-Cre
(Cdc42 cultures) or EGFP/ tdTomato alone (control cultures; for
control of Cre toxicity, see Materials and Methods). Two weeks
after transduction, control and Cdc42 cultures were stained for
Cdc42 (Fig. 2 D–E). Cre-transduced cells lacked specific staining
(Fig. 2 E–E), thereby confirming that the Cdc42 gene was
success-
fully deleted and Cdc42 protein levels were substantially re- duced
after lentiviral transduction.
After wounding the confluent astrocyte monolayer (for exper-
imental design, see Fig. 3A), the reaction of astrocytes was fol-
lowed in control and Cdc42 cultures. As expected, most of the
astrocytes lining the scratch in control cultures formed long po-
larized protrusions during the first 24 h (Fig. 3B). In contrast,
transduced astrocytes in Cdc42 cultures appeared less orga- nized,
with multiple protrusions extending randomly from cells (Fig.
3C–E).
To examine the development of this effect more quantita- tively, we
defined protrusions as (1) cell extensions that were at least three
times longer than wide and (2) oriented into the cell- free
scratch. We then assessed their appearance at different times after
injury. Cells were scored as “unipolar protruding” when they formed
a protrusion into the scratch without obvious exten- sions into
other directions. After 30 min, only a small percentage of control-
or Cre-transduced astrocytes had formed a protrusion into the
scratch (7 0.8% of control-transduced cells with pro- trusion 0.5 h
p.i., n (cultures) 6). Over time, an increasing number of
control-transduced cells formed protrusions into the cell-free
area, and at 24 h p.i., more than half of the cells were clearly
elongated toward the injury site (55.2 2.4% control- transduced
cells with protrusion 24 h p.i., n 6). In contrast, significantly
fewer Cre-transduced Cdc42 cells formed unipolar protrusions
oriented into the scratch at this time (21.6 3.0% Cre-transduced
cells with protrusion 24 h p.i., n 6, p 0.0001). In addition to
this significant reduction of Cdc42 unipolar cells with scratch
oriented protrusions we also noted many Cdc42
Figure 2. Localization of Cdc42 and protein loss after gene
deletion. A, Cdc42 protein is distributed around the nuclei of
cells shortly after scratching the monolayer. B, C, Eight hours
later, Cdc42 is relocalized toward the leading edge (B) and to the
tips of tubulin filaments (C). C–E, Two weeks after transduction
with control- or Cre-virus, Cdc42 protein expression was examined.
Lower-magnification pictures of not completely confluent cultures
show diffuse Cdc42 staining and enrichment of the protein as dots
at cell borders and within the cells in control-transduced and
nontransduced cells (D–D). In contrast, Cdc42 was absent in all
these places in tomato-IRES-Cre transduced cells (E–E). Strong red
fluorescence around the nuclei of transduced cells breaking through
into the green channel could be observed in control and Cdc42
cultures. Since this effect was also observed in live imaging
experiments, it appears to be intrinsic clustering of the tdTomato
protein in the Golgi compartment.
12474 • J. Neurosci., August 31, 2011 • 31(35):12471–12482 Robel et
al. • Cdc42 Is Important for Astrocyte Recruitment to Injury
unipolar cells with protrusions oriented parallel or even away from
the scratch (see example in Fig. 3C) as well as cells with multiple
protrusions (see example in Fig. 3D). Indeed, signifi- cantly more
Cdc42 cells had a higher number of protrusions than control cells
(Fig. 3E), clearly demonstrating that the re- duced number of
unipolar cells orienting toward the scratch is not due to a failure
of process formation. We therefore asked whether this defect might
be due to defects in polarization.
Previously, it has been shown that astrocytes place their MTOC in
front of their nucleus toward the direction of a scratch injury,
and this appears to be a prerequisite for oriented protru- sion
formation (Etienne-Manneville and Hall, 2001). To investi- gate
whether the reorientation of the MTOC was disturbed, we used the
same assay (Fig. 3A) and compared the number of re-
oriented MTOCs in control- and Cre- transduced astrocytes (Fig.
3F–H). Since the MTOC is located close to the nucleus, the area
around the nucleus was separated into 4 equal quadrants and placed
such that one quadrant was facing the scratch with each 90° angle
being either perpen- dicular or parallel to the scratch (Fig. 3G).
In nonoriented cells, the MTOC should be located randomly around
the nucleus, i.e., in 25% of all cases in any of the 4 quadrants.
Only cells with MTOCs clearly belonging to a given nucleus were
included in the quantification, and they were scored as reoriented
when they were located in the quadrant facing the scratch (Fig.
3G).
At 30 min after wounding MTOCs were facing the scratch in a random
man- ner. As soon as 4 h p.i., some control- transduced astrocytes
adjacent to the scratch started to reorient their MTOC to- ward the
scratch (data not shown). This proportion increased even further at
24 h p.i. (Fig. 3F,G). Comparable to control cells, at the start of
the experiment, MTOCs of Cdc42 astrocytes were ran- domly facing
the scratch area. However, at 24 h p.i., the number of reoriented
MTOCs within Cdc42 astrocytes did not increase further (Fig.
3F,H–H), indicat- ing that Cdc42 is required for MTOC ori- entation
toward the scratch.
Loss of Cdc42 causes impaired migration after scratch injury in
vitro The above data suggest that Cdc42 dele- tion leads to defects
in the initial orienta- tion of astrocytes toward the scratch.
However, as these data were obtained in fixed cultures, we next
used time-lapse video microscopy to observe protrusion formation
dynamics in relation to cell mi- gration of virally transduced
cells over several days (Fig. 4A).
As expected, control-transduced as- trocytes and nontransduced
astrocytes adjacent to the scratch formed unipolar protrusions,
translocated their cell bodies,
and retracted their rear sides to migrate into the scratch. Within
5 d, astrocytes in control cultures had completely closed the 500 m
wide scratch (Fig. 4B; Movie 1). However, Cdc42 astrocytes migrated
virtually randomly and were often overtaken by WT cells (Fig. 4B;
Movie 2). To clarify the causes for these defects after loss of
Cdc42, we examined astrocyte migration and focused on protrusion
formation, stability, and orientation, as well as nu- clear
translocation, as these are all crucial steps in cell migration and
scratch wound closure.
Consistent with the data from still analysis described above,
protrusion formation per se was not impaired in Cdc42 astro- cytes
compared with control cells (66 6% of control- transduced cells and
72 4% of Cre-transduced cells formed protrusions 24 h p.i., n 3),
while protrusion orientation was re-
Figure 3. Cdc42 is involved in orienting protrusions and the MTOC
toward a scratch wound in vitro. A–D, Astrocytes of postnatal
Cdc42fl/fl mice were cultured in a monolayer, transduced with
Cre-IRES-tdTomato or tdTomato virus and scratch wounded 2 weeks
later (A). In control cultures most cells formed long unipolar
protrusions toward the direction of the scratch (B). Cdc42 cultures
were characterized by misoriented cells (white arrowhead in C) and
many cells with multiple protrusions directed into various
directions around the cell body (D). E, Quantification of the
number of protrusions per cell is shown (this quantification was
done according to the experimental scheme in Fig. 3A at 24 h p.i.).
F, Quantification of MTOC reorientation 24 h p.i. shows a
significant reduction in MTOC reorientation after deletion of
Cdc42. G–H, The majority of first row astrocytes had MTOCs oriented
toward the scratch (G, G, G), whereas MTOCs of Cre-transduced cells
were randomly located around the nucleus (H, H, H). To quantify
MTOC orientation, the nucleus was divided into 4 quadrants and
MTOCs located in the quadrant facing the scratch were scored as
oriented (G). P5, Postnatal day 5.
Robel et al. • Cdc42 Is Important for Astrocyte Recruitment to
Injury J. Neurosci., August 31, 2011 • 31(35):12471–12482 •
12475
markably different in Cdc42 astrocytes that had a higher number of
protrusions that were randomly oriented compared with control cells
(Fig. 3C–E; data not shown). To understand the cause for the in-
crease in protrusion number in Cdc42 as- trocytes, we examined
protrusion turnover. Within the first 24 h p.i., protrusion turn-
over was comparable between Cdc42 and control astrocytes (Fig. 4C).
Thereafter, the number of instable protrusions per cell de- creased
significantly in control astrocytes, due to stabilization of
previously formed protrusions. This was not the case for Cdc42
astrocyte protrusions, which re- tained a higher turnover rate at
48 h p.i. (Fig. 4C). Thus, Cdc42 astrocytes have difficul- ties in
stable maintenance of protrusions over time.
Since defects in process maintenance may affect migration, we next
tracked nu- clei of control- or Cre-transduced cells over a period
of 5 d with hourly distance measurement (132 data points) depicted
in a tracking path (Fig. 4D). A starting position and an end
position was defined for three different time points (1, 3, 5 d
p.i.), and based on the fluorescent images taken each hour, the
software performed automated tracking. As evidenced by the examples
shown in Figure 4D, the track- ing paths of control astrocytes had
a straight linear appearance, whereas the majority of Cdc42 cells
took a rather coiled path (Fig. 4D). Consistent with this
impression, the straight distance migrated (shortest path from the
starting position to the end position, Fig. 4E) was signifi- cantly
reduced for Cdc42 astrocytes to virtually half of the straight
distance cov- ered by control cells over the same period (Fig. 4
F). Conversely, the total migra- tion distance, represented by the
overall migration distance of a cell including forward, backward,
and sideward move- ments (Fig. 4E), was comparable between control
and Cdc42 astrocytes (Fig. 4G). Consistent with the equivalent
migration distance between control and Cdc42 cells, the average
velocity was also not sig- nificantly different between control and
Cdc42 cells at 1, 3, and 5 d p.i. (Fig. 4H). In summary, the
overall ability of Cdc42 cells to migrate was not impaired, but
directed migration toward the scratch was aberrant.
If cells migrate the same total distance at the same speed, but
cover less straight distance, their migration pathway would likely
be rather coiled and curved. This was measured as the tortuosity,
the quotient of total and straight distance. An absolute linear
movement in one direction (with identical straight and total dis-
tance) would have a tortuosity value of 1. The tortuosity of
control-infected astrocytes was 2.5 0.3, i.e., their path was 2.5
times longer than a direct route from start to end. Cdc42
cells
exhibited continuously increased tortuosity values from day 1 (2.6
0.2) to day 5 (4.4 0.6) that reached almost double the tortuosity
values of control cells (Fig. 4 I). Thus, loss of Cdc42 in
astrocytes resulted in significantly increased directional changes,
despite an overall equal capacity for migration as reflected in the
comparable total migration distance and velocity.
The role of Cdc42 in astrocytes at a stab wound injury in vivo
These results demonstrate that Cdc42 astrocytes can extend
protrusions and migrate at normal speed, but they do so in an
undirected manner that ultimately impairs wound closure in
Figure 4. Reactive astrocytes lacking Cdc42 show abnormal migration
behavior in vitro. A, The migratory behavior of Cdc42fl/fl
astrocytes transduced with lentiviral particles encoding
tdTomato-IRES-Cre or tdTomato alone was analyzed using the in vitro
scratch wound assay combined with time-lapse video microscopy.
Movie gallery (3 channel overlap: phase contrast; blue, Hoechst
live dye; red, tdTomato reporter) of the progression of wound
closure by scratch-activated astrocytes 0, 1, 2, 3, and 5 d p.i. B,
Nontransduced and control-transduced cells filled the scratch
within 5 d, while Cdc42 cells showed migration deficits. C, Quan-
tification of the protrusion turnover rate revealed an increase in
instable protrusions in Cdc42 cells. D, Migration paths recorded by
tracking the nuclear translocation over 5 d, show disoriented
movements of Cdc42 astrocytes when compared with control cells. E,
Schematic representation of total migration distance (nuclear path)
and straight distance (direct route); both parameters were measured
for transduced astrocytes at the scratch 1, 3 and 5 d p.i. F–H,
Analysis of the tracking data revealed a reduction of the straight
nuclear migration distance in the Cdc42 culture 3 d p.i and later
(F ), but no difference in total migration distance (G) and mean
velocity (H ) between control and Cdc42 cells. An increase in
tortuosity at 3 and 5 d p.i. further confirmed the orientation
defect of migrating Cdc42 astrocytes (I ).
12476 • J. Neurosci., August 31, 2011 • 31(35):12471–12482 Robel et
al. • Cdc42 Is Important for Astrocyte Recruitment to Injury
vitro. This raises the question of whether the defects observed in
Cdc42 astrocytes in the scratch wound assay in vitro can be
observed in vivo, where astrocytes react to a complex milieu of
signals released by multiple cell types. To examine the behavior of
Cdc42 astrocytes in vivo, we used the stab wound lesion model in
the adult mouse cerebral cortex (Buffo et al., 2005, 2008), and
monitored the polarity reaction and recruitment of astrocytes
toward the site of this acute traumatic injury.
Astrocytes also reacted in vivo to injury by altering their mor-
phology assuming a bipolar shape within 7 d p.i. (compare Fig.
5A,B). To examine the full morphology of protoplasmic astro- cytes
beyond their GFAP processes (for differences between
GFAP-immunostaining and fully cytoplasmic extensions, see
Wilhelmsson et al., 2006), an EGFP reporter mouse line was crossed
to the Tamoxifen-inducible GLAST::CreERT2 line, which allows the
induction of genetic recombination in astro- cytes (Mori et al.,
2006; Buffo et al., 2008). Protoplasmic astro- cytes in the gray
matter of the cerebral cortex normally possess many fine, radially
arranged processes (Fig. 5C). After stab wound injury however, many
EGFP-labeled astrocytes became elongated and extended long
processes toward the injury border at 7 d p.i. (Fig. 5B,D). This
reaction was reminiscent of the “pal- isading zone,” a defined
region next to the injury core described previously in mouse models
of epilepsy (Oberheim et al., 2008). After stab wound, elongated
astrocytes were only detected within
an approximate area of 200 m around the lesion site, while further
away, reactive astrocytes retained their radial symmetry and did
not become polarized (Fig. 5E). As observed by GFAP-immunostaining
(Fig. 5A,B), also analysis of full morphol- ogy revealed that the
polarity reaction and formation of the palisading zone devel- oped
gradually with few astrocytes begin- ning to elongate and extending
processes toward the injury border at 3 d p.i., while more than one
third of reactive astro- cytes proximal to the injury border had an
elongated and polar morphology with long processes oriented toward
the injury site at 7 d p.i., Figs. 5D, 6).
To examine the role of Cdc42 in polar- ization of astrocytes toward
the injury site in vivo, Cdc42 was conditionally deleted in
astrocytes in the adult brain using the GLAST::CreERT2 mouse line
crossed to the above described line with loxP sites flanking exon 2
of the Cdc42 gene. Re- combination was achieved by administra- tion
of the estrogen analog Tamoxifen to 2- to 3-month-old mice
heterozygous for GLAST::CreERT2, positive for the EGFP- reporter,
and homozygous (referred to as Cdc42), heterozygous, or-negative
(referred to as control) for the floxed Cdc42 allele. Four weeks
after Tamoxifen administration, when Cdc42 protein should be
largely gone, a stab wound was placed in the gray matter of the
cerebral cortex (Fig. 6A). First, we examined ex- pression of GFAP,
an intermediate fila- ment characteristically upregulated in
parenchymal astrocytes in response to in-
jury. As expected, a high number of recombined astrocytes close to
the injury site expressed GFAP in control animals (93.5 2.0 GFAP
EGFP cells among EGFP, n (animals) 3; Fig. 6B). After deletion of
Cdc42, a comparable number of astrocytes up- regulated GFAP (95.9
1.7% GFAP EGFP cells among EGFP in Cdc42, n 3, p 0.43; Fig. 6C) and
showed a hyper- trophic morphology, suggesting that overall
injury-induced reac- tivity was not disturbed by the loss of
Cdc42.
Next, we examined the polarization of astrocytes by quantifying
cells that had formed an elongated protrusion at 7 d p.i., when the
palisading zone was well established in the control. Accordingly,
39.2 1.7% (n 3) of the EGFP control cells in the palisading zone
had formed a protrusion oriented toward the injury (Fig. 6D).
Surprisingly, this number was significantly enhanced in Cdc42 as-
trocytes (68.4 3.6%, n 4, p 0.001; Fig. 6E). Cdc42 astrocytes were
more elongated (83.3 6.8, n 5), with a significant increase in
total length compared with control astrocytes (57.6 6.3 m in
control, n 3, p 0.044; quantified according to the panel depicted
in Fig. 6F). This was an effect of the stab wound injury, as no
differ- ences in astrocyte size or morphology were observed in the
contralat- eral hemispheres (data not shown). Thus, in sharp
contrast to the in vitro response, the change toward a bipolar
morphology is even more pronounced in astrocytes lacking
Cdc42.
In response to injury, astrocyte number increases around the lesion
site (Sofroniew and Vinters, 2010). Given that Cdc42-
Movie 1. Astrocyte polarization and migration after in vitro
scratch wound. Scratch-wounded astrocyte monolayer, followed over 3
d by time-lapse video microscopy. Nontransduced cells and
control-transduced cells (expressing the red fluorescent protein
tdTomato) polarize perpendicular to the scratch, and thereafter
migrate into the cell-free cleft to fill up the wound.
Movie 2. Astrocytes lacking Cdc42 show deficits in polarized
migration. After scratching an astrocyte monolayer, Cdc42-
deficient cells (expressing the red fluorescent protein tdTomato)
show impaired scratch-directed polarization and migration.
Wild-type cells (cells that do not express tdTomato) bypass
impaired Cdc42 astrocytes.
Robel et al. • Cdc42 Is Important for Astrocyte Recruitment to
Injury J. Neurosci., August 31, 2011 • 31(35):12471–12482 •
12477
deficient astrocytes showed impaired directed migration in vitro,
we asked whether astrocyte recruitment toward the injury site in
vivo would also be affected. We quantified the number of EGFP cells
in the hemisphere contralateral to the injury to control for
recombination effi- ciency, and observed an equal number of cells
in control and Cdc42 brains (99.7 17.8% of recombined cells in
Cdc42 brains, n 8, relative to recom- bined cell number in control
brains, n 6, p 0.99), demonstrating equal recombination rates.
However, within the palisading zone around the stab wound (0 –100 m
from the injury core) the number of Cdc42 EGFP cells was re- duced
to less than half (236.8 51.1 cells per mm 2 in control, versus
95.5 9.2 in Cdc42, n 4, p 0.0347), suggesting a severe defect in
astrocyte recruitment to- ward the injury site in the absence of
Cdc42.
Astrocyte-specific loss of Cdc42 leads to increased microglia
number at the stab wound injury in vivo Notably, while we observed
a strong de- crease in the proportion of recombined astrocytes at
the injury site, only approxi- mately one third of all astrocytes
were re- combined in both controls and fl/fl mice (27.5 2.7% in
control 25.9 4.8% in Cdc42, n 3, p 0.78). We then considered
whether even such a small 15% decrease in the total population of
reactive astrocytes at the injury site might be sufficient to
affect other cell types surrounding the injury site. Microglia are
the resident immune cells of the brain and are activated and
recruited toward injury, most likely interacting with astrocytes
throughout reactive gliosis (Hanisch and Kettenmann, 2007). To
understand whether the reaction of microglia to injury was changed
after loss of Cdc42 in the recombined astrocytes at 7 d p.i., we
quantified Iba1-positive microglia. Contralateral to the injury
site, the number of micro- glia was similar between control and
Cdc42 brains (9023 1494 Iba1 cells per mm 3 in control and 7916 665
Iba1 cells per mm 3 in Cdc42, p 0.54; Fig. 7A,B). As expected, the
number of microglia dramatically increased directly at the lesion
(Fig. 7C,D). In the control, microglia number relative to the con-
tralateral hemisphere was approximately fivefold higher at a dis-
tance of 100 –250 m from the injury site and tenfold higher
directly at the injury site (0 –100 m) (Fig. 7C,E). This increase
was even more pronounced after astrocyte-specific deletion of
Cdc42. Here, a 12.5-fold increase in microglia was observed (Fig.
7D,E; n 3, p 0.031). Interestingly, the increase in microglia
number was observed precisely in the region where astrocyte numbers
were decreased (see above), but not at further distant sites (Fig.
7E). Thus, even though only a subset of astrocytes was affected in
recruitment to the injury site, these changes were suf- ficient to
affect the microglia reaction.
The proper reaction of astrocytes and microglia postinjury is
thought to be essential for protection of the brain from primary
neuronal loss. Since both of these cell types are changed after
loss of Cdc42, we next examined neuronal number at the injury
site
(Fig. 7F). The pan-neuronal marker NeuN is typically downregu-
lated in neurons surrounding the injury site (data not shown),
therefore we used cresyl violet for neuronal somata detection (see
red arrow in Fig. 7G,H; Fig. 7G, inset) and compared neuronal cell
number in close proximity to the injury site to a similar brain
region at 500 m distant from the injury. Notably, neuron number was
reduced to approximately one-third within 100 m around the stab
wound at 3 and 7 d p.i. (n 6, Fig. 7F–H), but at 100 –200 m distant
from the injury, their number was compa- rable to far distant
regions (93.7 13.2% neurons in control, 82.5 9.4% neurons in brains
with Cdc42 astrocytes, normal- ized to neuronal number distal to
the injury site, n 3, p 0.53), indicating a rather concise region
of neuronal death in close vi- cinity to the injury site. In brains
with recombined astrocytes depleted of Cdc42 (Fig. 7G), neuron
number was comparably reduced to within 100 m of the injury site at
3 or 7 d p.i. (Fig. 7F,H, n 8, p 0.48). This is consistent with a
comparable number of apoptotic cells detected by TUNEL, 3 d p.i.
(9685 4634 TUNEL cells per mm 3 in control brains, 7277 1490 TUNEL
cells per mm 3 in brains with Cdc42 astrocytes, n 6, p 0.63),
indicating that primary neuronal death in response to injury is not
affected by the modest reduction of astrocyte recruitment achieved
by inducible Cdc42 deletion in 30% of adult astrocytes.
Discussion Here, we demonstrate an essential role for the small
RhoGTPase Cdc42 for recruitment of astrocytes to an injury site in
vitro and in vivo. While injury-oriented process formation was
impaired2 in the absence of Cdc42 in vitro, it appeared normal in
vivo. In
Figure 5. Astrocytes change their morphology after acute injury in
vivo. A, B, After stab wound injury, GFAP astrocytes were clearly
hypertrophic at 3 d p.i. (A). At 7 d p.i. astrocytes formed a
palisading zone directly around the lesion (B). The red line
outlines the lesion core in A and B. C, EGFP labeling of single
astrocytes revealed their morphology in greater detail. Gray matter
astrocytes in the intact cerebral cortex have a star-like
morphology with few main processes that ramify into many fine
branches. D, E, At 7 d p.i., 2 types of reactive astrocytes were
observed. D, Directly at the lesion site astrocytes extended a few
thick processes toward the injury site. E, Distal from the injury
site GFAP, reactive astrocytes were not elongated. The white line
in D indicates the lesion site.
12478 • J. Neurosci., August 31, 2011 • 31(35):12471–12482 Robel et
al. • Cdc42 Is Important for Astrocyte Recruitment to Injury
contrast, the increase in astrocyte number at the injury site could
not be compensated for in vivo. Most importantly, even a modest
(based on the recombination frequency of 30%) reduction in
astrocyte recruitment to the injury site resulted in a significant
increase in microglia number at the injury site, suggesting a cru-
cial role of astrocytes in reducing microglia number at the injury
site.
Polarity and migration of astrocytes after injury in vitro Scratch
injury in vitro is a well established assay used to monitor
directed cell migration. Astrocytes in vitro polarize toward a
scratch by positioning the centrosome/MTOC between their nu- cleus
and their leading edge and forming directed protrusions before
migration into the cell-free scratch (Etienne-Manneville and Hall,
2001, 2003; Etienne-Manneville et al., 2005; Holtje et al., 2005;
Etienne-Manneville, 2006; Peng et al., 2008; Ang et al., 2010).
Consistent with previous experiments using dominant- negative
(Dn)Cdc42, genetic deletion of Cdc42 in astrocytes in vitro
resulted in MTOC misorientation and a decreased number of cells
exhibiting scratch oriented unipolar protrusions, thereby
supporting the idea that Cdc42 affects astrocyte polarity in vitro
(Etienne-Manneville and Hall, 2001; Etienne-Manneville,
2008a,b; Li and Gundersen, 2008; Bartolini and Gundersen, 2010).
However, we could not confirm all the defects previously observed
after DnCdc42 (Etienne-Manneville and Hall, 2001; Czuchra et al.,
2005); for example protrusion formation was un- disturbed after
genetic deletion of Cdc42 in astrocytes in vitro and in vivo.
Conversely, Cdc42 cells often appeared multipolar with
multidirectional protrusions around the cell body soon after the
scratch. This discrepancy could be due to the dominant-negative
constructs affecting other RhoGTPases, since they bind to corre-
sponding guanine nucleotide exchange factors (GEFs) with a higher
affinity than endogenous RhoGTPases, preventing effec- tor
interaction and subsequent signaling (Feig, 1999). As GEFs are
often shared by several RhoGTPase members (Schmidt and Hall, 2002;
Rossman et al., 2005), DnCdc42 may also affect Rac1, which is
localized to the leading edge of scratch-activated cells by
Cdc42-dependent Pak activity, and is responsible for protrusion
formation (Cau and Hall, 2005).
Cell migration is governed by the ability to extend, retract, and
stabilize membrane protrusions in a defined direction. This can
occur in a noncoordinated manner, resulting in random migra- tion,
or in a coordinated manner, resulting in directed migration in
response to environmental cues (Etienne-Manneville, 2008a). Indeed,
tracking Cdc42 astrocyte nuclei revealed that overall migration was
not impaired. However, their tracking paths into the scratch were
coiled showing that their directionality was lost. We conclude that
in Cdc42 astrocytes an initial polarization defect leads to
randomly oriented MTOCs that subsequently cause disoriented
movement.
Defects in astrocyte recruitment to the site of brain injury after
Cdc42 deletion in astrocytes of the adult brain Here, we unravel a
hitherto unrecognized role of the small RhoGTPase Cdc42 in
astrocyte recruitment to the injury site in vivo, without affecting
overall astrocyte reactivity (Okada et al., 2006; Herrmann et al.,
2008), since GFAP upregulation and hy- pertrophic response after
injury were normal. Interestingly, in contrast to what has been
found in vitro, the polarity reaction of astrocytes in the
palisading zone adjacent to the injury site was not impaired by
Cdc42 deletion, but even enhanced with more cells elongated toward
the injury. This discrepancy highlights the limitations of the in
vitro scratch assay and the complex nature of cellular interactions
and multiple signaling pathways after injury in vivo. While
astrocytes in the scratch wound assay are exposed to a cell-free
scratch, and almost exclusively astrocyte-released autocrine
signals, astrocytes are exposed to a much larger reper- toire of
signals released from a multitude of cells in vivo, includ- ing
degenerating neurons, oligodendrocytes and their progenitor cells,
the NG2 glia, microglia, and invading cells from the blood system.
Indeed, we found that microglia numbers were signifi- cantly
increased surrounding the stab wound site, thus possibly
representing a source of additional signals mediating orientation
of palisading astrocytes toward the injury site. Therefore, the in
vitro assay is well suited to examine cell-autonomous effects, but
extrapolation to the in vivo situation may not always be
possible.
Mechanisms controlling Cdc42 activation and localization to the
leading edge of the cell are still poorly understood, but ADP
ribosylation factor 6 (Arf6)-dependent membrane traffic is such a
crucial factor for recruitment of Cdc42 to the leading edge (Osmani
et al., 2010). Moreover, Cdc42 is a downstream effector of integrin
signaling (Etienne-Manneville and Hall, 2001; Os- mani et al.,
2006; Etienne-Manneville, 2008b). Interestingly, in- terference
with 1-integrin-mediated signaling at postnatal stages by genetic
deletion results in reactive astrogliosis even in
Figure 6. The effects of Cdc42 deletion in astrocytes on their
morphology at the injury site in vivo. A, Genetic recombination was
induced in 2- to 3-month-old animals that were stab wound injured 4
weeks later and killed 7 d p.i. following the schedule in A. B–E,
Astrocytes at the injury site strongly upregulated GFAP in control
(B) and Cdc42 (C) brains. In control brains,40% of recombined EGFP
astrocytes formed a protrusion (white arrows, nonprotruding cells
are highlighted by a white arrowhead) within the palisading zone
(D). This number was increased in Cdc42 animals (E). F,
Measurements of protrusion and cell length were done.
Robel et al. • Cdc42 Is Important for Astrocyte Recruitment to
Injury J. Neurosci., August 31, 2011 • 31(35):12471–12482 •
12479
the uninjured brain in vivo (Robel et al., 2009), and interference
with integrin sig- naling in astrocytes in vitro blocks protru-
sion formation and polarity (Etienne- Manneville and Hall, 2001;
Osmani et al., 2006; Peng et al., 2008). Notably, in vivo,
palisading zone formation and bipolar orientation could also occur
in the ab- sence of 1-integrins in astrocytes (data not shown),
further supporting the con- cept of alternative pathways in
astrocyte orientation in vitro (requiring 1-integ- rins and Cdc42)
and in vivo (not requiring either of these). However, other
integrins may be compensating in the absence of Cdc42 to mediate
effects on astrocyte po- larity via other effector pathways (Holly
et al., 2000; Lemons and Condic, 2008). For example, 64 integrins
interact with in- termediate filaments (Rezniczek et al., 1998),
which are strongly upregulated af- ter brain injury in astrocytes
and may play a key role in stabilizing palisading bipolar
astrocytes at the site of injury in vivo. In addition, the basement
membrane recep- tor dystroglycan has been shown to be necessary for
astrocyte polarization (Peng et al., 2008), and could act as a
redundant mechanism for reactive astrocyte polar- ization in
vivo.
Although Cdc42 astrocytes were po- larized in vivo, the increase in
astrocyte number surrounding the injury site was severely impaired,
with less than half of the recombined Cdc42-deficient astro- cytes
found at the injury site. This is not due to developmental defects,
as Cdc42 was deleted in fully mature astrocytes in the adult brain
by Tamoxifen-mediated recombination using GLAST::CreERT2 mice (Mori
et al., 2006; Buffo et al., 2008). We therefore conclude that Cdc42
plays a specific and non-redundant role after brain injury in
regulating astrocyte re- cruitment to the lesion site. Most impor-
tantly, recruiting fewer astrocytes to the injury site also affects
another cell type as detailed below. It will therefore be impor-
tant to unravel the precise mechanisms of Cdc42-dependent astrocyte
recruitment in vivo. Both directed cell migration and proliferation
have been implicated in this process (Okada et al., 2006; Auguste
et al., 2007; Buffo et al., 2008; Sofroniew and Vinters, 2010), and
only live in vivo imag- ing will be able to directly determine
which of these processes is defective in the absence of
Cdc42.Figure 7. The effects of Cdc42 deletion in astrocytes on
microglia and neurons at the injury site in vivo. A–E,
Iba1-labeled
microglial cells are shown in brains with control (A, B) or Cdc42
(C, D) astrocytes 7 d p.i. There were comparable numbers of resting
Iba1 microglia in the contralateral hemispheres of control (A) and
Cdc42 (B) brains. The microglia number significantly increased
close to the injury site in brains with control (C) or Cdc42 (D)
astrocytes, but numbers were increased even further after deletion
of Cdc42 in astrocytes (E). F–H, Neurons were visualized by cresyl
violet staining as pale purple cells (G, H; indicated by red arrows
and enlarged in the inset in G), and stereotactic counting of these
revealed no significant difference after deletion of Cdc42 at the
injury site (F ). Neuronal numbers at the injury site were
normalized to numbers quantified in a distal unaffected
region
4
(F). Small or shrunken dark purple cells were excluded from the
quantitative analysis as they represent glial and/or dying cells
(see white arrowheads in H). sw, Stab wound.
12480 • J. Neurosci., August 31, 2011 • 31(35):12471–12482 Robel et
al. • Cdc42 Is Important for Astrocyte Recruitment to Injury
Consequences of reduced astrocyte recruitment after injury
Activated astrocytes contribute to scar formation not only by
increasing in number, but also by releasing a multitude of mole-
cules, such as chondroitin sulfate proteoglycans, cytokines, and
mitogens (Buffo et al., 2010) that act on other cell types. There-
fore, a key question was to what extent even a small change in the
number of recruited astrocytes may impact other cell types. In-
deed, reduction of half of all recombined astrocytes (15% of all
astrocytes), resulted in a significant increase in microglia number
at the injury site. These observations support quantitative signal-
ing between reactive astrocytes and microglia. Indeed, reactive
astrogliosis in the uninjured brain as elicited by 1-integrin dele-
tion (Robel et al., 2009) also affected microglial cells, and
astrocyte-conditioned medium has been shown to affect the state of
microglia activation (Schilling et al., 2001; Kim et al., 2010),
consistent with direct signaling from activated astrocytes to mi-
croglia. In addition, Cdc42-deficient astrocytes may be defective
in their release of signaling molecules due to possible alterations
in their secretory activity (Harris and Tepass, 2010). To reveal
the precise role of microglia in this context, it will be
interesting to investigate whether they are in a “beneficial” state
(Thored et al., 2009; Kettenmann et al., 2011) to compensate for
the reduction in astrocytes, or whether the increase in microglia
is an indicator of an increased detrimental inflammatory reaction
due to the defects in Cdc42-deficient astrocytes. Further analysis
of reactive astrocytes and microglial cells in this context will be
required to determine their exact activation and signaling state.
Thus, condi- tional deletion of Cdc42 in astrocytes will serve as a
useful model to further study interaction between glial cell types
in vivo with the aim of dissecting pathways eliciting the
beneficial or adverse roles. Beyond the precise mechanisms, this
analysis highlights the key role of reactive astrocytes at the
injury site and the profound effect of even small alterations in
their number.
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